Ca²⁺-mediated activation of the skeletal-muscle ryanodine receptor ion channel

Cryo-electron micrograph studies recently have identified a Ca²⁺-binding site in the 2,200-kDa ryanodine receptor ion channel (RyR1) in skeletal muscle. To clarify the role of this site in regulating RyR1 activity, here we applied mutational, electrophysiological, and computational methods. Three amino acid residues that interact directly with Ca²⁺ were replaced, and these RyR1 variants were expressed in HEK293 cells. Single-site RyR1-E3893Q, -E3893V, -E3967Q, -E3967V, and -T5001A variants and double-site RyR1-E3893Q/E3967Q and -E3893V/E3967V variants displayed cellular Ca²⁺ release in response to caffeine, which indicated that they retained functionality as caffeine-sensitive, Ca²⁺-conducting channels in the HEK293 cell system. Using [³H]ryanodine binding and single-channel measurements of membrane isolates, we found that single- and double-site RyR1-E3893 and -E3967 variants are not activated by Ca²⁺ We also noted that RyR1-E3893Q/E3967Q and -E3893V/E3967V variants maintain caffeine- and ATP-induced activation and that RyR1-E3893Q/E3967Q is inhibited by Mg²⁺ and elevated Ca²⁺ RyR1-T5001A exhibited decreased Ca²⁺ sensitivity compared with WT-RyR1 in single-channel measurements. Computational methods suggested that electrostatic interactions between Ca²⁺ and negatively charged glutamate residues have a critical role in transducing the functional effects of Ca²⁺ on RyR1. We conclude that the removal of negative charges in the recently identified RyR1 Ca²⁺-binding site impairs RyR1 activation by physiological Ca²⁺ concentrations and results in loss of binding to Ca²⁺ or reduced Ca²⁺ affinity of the binding site.